The endosomal sorting complex required for transport-III (ESCRT-III) is an ancient and highly conserved membrane remodeling machinery that catalyzes a wide range of cellular membrane remodeling processes, including the budding of enveloped viruses such as HIV-1. Due to the spatial resolution and transient nature of ESCRT-III assembly in vivo, understanding the architecture of the native ESCRT-III complex at HIV-1 budding sites remains challenging.
​ In this thesis, we developed a drug-inducible transient inhibitory tool to enhance the ESCRT-III lifetime at HIV-1 budding sites. We generated CHMP4B, CHMP3, and CHMP2A fusion proteins with the hepatitis C virus NS3 protease separated by a linker containing the protease cleavage site, which allows autocleavage upon expression. Then we characterized the CHMP-NS3 fusion proteins in the absence and presence of the protease inhibitor Glecaprevir with respect to expression, stability, localization and HIV-1 Gag VLP release.
Western blotting analysis revealed rapid and stable accumulation of CHMP-NS3 fusion proteins with a dominant-negative effect on Gag VLP release upon drug treatment ; CHMP4B-NS3 and CHMP2A-NS3 fusion proteins reduced VLP release, whereas CHMP3-NS3 exerted a minor effect and synergize with CHMP2A. Localization studies demonstrated the re-localization of dominant-negative CHMP-NS3 fusion proteins to the plasma membrane, endosomes and Gag VLP budding sites. Transmission electron microscopy imaging confirmed the drug-dependent impairment of VLP release with VLPs connected to the plasma membrane via membrane necks. In addition, the impairment of VLP release upon Glecaprevir addition is characterized by an extended retention of VLPs at the plasma membrane in the presence of CHMP4B-NS3 and CHMP2A-NS3.
Overall, our findings provide insights into the functional consequences of inhibiting ESCRT-III-mediated membrane fission during HIV-1 budding, and highlight the potential of a novel drug-inducible tool for studying the native ESCRT architecture at HIV-1 budding sites or other ESCRT-catalyzed cellular processes.

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